Molecuiar Microbioiogy (1990) 4(7), 1165-1172
Isolation and characterization of the recA gene of Bordetella pertussis S. A. Kuhl,* R. P. McCreary,^ J. D. Bannan and R. L. Friedman Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, Arizona 85724, USA. Summary This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coii RecA" mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA~ strains to tolerate the DNA-damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5kb 5ful-£coRI fragment, and expression of the gene in E. coli resulted in the production of a 40 kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA-like activity has been isolated and cloned.
Introduction The recA gene is one of a number of genes termed 'housekeeping genes' that are concerned with maintenance of cellular functions. The RecA protein has been extensively studied in Escherichia coli, and its dual regulatory roles in the 'SOS-response' Induced by DNA-damaging agents (Witkin, 1976; Little and Mount, 1982; Walker, 1984; 1985) and homologous recombination (Smith, 1988) have been reviewed recently. Researchers have reported (Little, 1984; Slilaty and Little, 1987) that the RecA protein's role in regulating the SOS-response (to DNA damage) is to bind to the LexA repressor protein and stimulate its autodigestion. Cleavage of the LexA repressor derepresses a large number of operons concerned with coping with DNA damage (Little and Mount, 1982; Walker, 1984; 1987; Peterson et al., 1988). The RecA protein also promotes the cleavage of prophage
Received 22 November, 1989. tPresent address: Department of Biology, US Air Force Academy, iJSAFA/DFB, Colorado Springs, Coiorado 80840, USA. 'For correspondence, Tei, (602) 626 6061; Fax (602) 626 2100.
repressors (Roberts and Roberts, 1975; Roberts et al., 1977; Little, 1984), allowing lytic grovrth of bacteriophage to be initiated. Studies performed on a number of different bacteria, including E co//(Walker, 1984; Kokjohn and Miller, 1985; Smith, 1988; Setlow efa/., 1988), have indicated that the recA gene product is required for induced mutagenesis caused by u.v. light and chemical DNA-damaging agents, recombination proficiency, and induction of some prophage. The E. coli recA gene has been cloned, a restriction map constructed (Sanoar and Rupp, 1979), and the DNA sequence determined by Horii etal. (1980) and Sancar et ai. (1980). These authors have found that the gene is 1059 nucleotide residues long and codes for a 38 kD protein. The recA gene has also been cloned from numerous different bacteria including both Gram-negative and Gram-positive organisms (Smith, 1988). One of our research Interests concerns the study of the genetic and molecular aspects of Bordetella pertussis pathogenesis. B. pertussis is a human pathogen that causes whooping cough, an acute and chronic respiratory disease that primarily affects young children (Linnemann, 1978). The virulence-associated and virulence-regulated genes of this organism have been the subject of many studies (Stibitz et al., 1988; Weiss and Falkow, 1984; Weiss et al., 1983), but few investigations have been performed on the regulation and expression of B. pertussis housekeeping genes. This paper describes studies concerned with the B. pertussis recA gene. It details the isolation and initial characterization of the DNA fragment containing this gene from a genomic library. The data presented demonstrate that the recA gene complements the defective RecA phenotype of £ co//recA" mutants by restoring resistance to the effects of DNA-damaging agents, thus confirming its identity.
Results Isolation of the B. pertussis reoA gene The purpose of this study was to isolate and clone the recA gene from 6. pertussis. E. coli strain DH5a(recA1) is sensitive to MMS-induced DNA damage, and can be used to select for clones that complement the RecA" phenotype. A genomic library of S. pertussis DNA in plasmid pUC18 transformed Into DH5a was spread onto agar
1166
S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L. Friedman
12 3 4 5 6 7
10
11
12
13
-23.1 -9.4 -6.6 -4.4
2.3 2.0
Fig. 1. Autoradiograph resulting from Southern hybridization of the 830bp Pst\ internal fragment of the pSAKIOI insert with plasmid DNA isolated from MMS-resistant clones. Plasmid DNA from the clones was digested with either f ^ f I (lanes 1-7) or EcoRI and H/ndlll (lanes 8-12) and subjected to agarose gel electrophoresis on an 0.8% agarose gel. The DNA fragments were transferred to Gene Screen Plus and hybridized to pP]-h6xanucleotic)e-labelled 830bp Psfl pSAKIOI internal fragment DNA. Lanes: 1 and 7. pUC18; 2 and 8, pSAKlOO: 3 and 9, pSAK101;4and10, pSAK102;5and11,pSAK104;6and 12, pSAK105; 13, H//1 dill-digested lambda D f ^ markers.
Hybridization ot plasmid pSAKIOI with genomic DNA from different Bordetella species With the recA gene from B. pertussis having been isolated, attempts were made to characterize the gene, using the plasmid with the smallest insert (pSAKI 01). To determine whether plasmid pSAKIOI (no Cla\ sites) would hybridize to a single unique band in Bcrdetelia strains, a ^^P-labelled probe of the pSAK101 insert was hybridized with Cla\digested genomic DNA isolated from strains of 6. pertussis, Bcrdetelia bronchiseptica, Bordetelia parapertussis. and Bordetelia avium. The autoradiograph (Fig. 2, lanes 1-6) clearly demonstrates that each of the S. pertussis, B. parapertussis, and B. brcnchiseptica strains examined possessed a single 8.4kb band, while the S. avium strains possessed a 5.9kb band that was homologous to the pSAKIOI piasmid (Fig. 2, lanes 7-8). Sinoe only a single homologous band was detected in B. pertussis genomic DNA, repetitive sequences are not associated with the B. pertussis recA gene, as was previously found in other regions of the 6. pertussis genome (McPheat and McNally, 1987; MoLafferty etaL, 1988; Park efa/., 1988).
Construction of a restriction map of pSAKIOI
plates supplemented with ampicillin and MMS to select for clones resistant to fVlMS (MMS'^. After 24h, MMS" colonies were found at a frequency of 1.69 x 10"^. When DH5a transformed with pUCI 8 without inserts was plated onto media containing MMS, no colonies were visible after 24h. Analysis of 10 representative colonies revealed that five different MMS'^ clones contained plasmids with different size inserts (pSAKlOO, 4.5kb; pSAKIOI, 3.5kb; pSAK102,5.4kb; pSAK104,6.0kb; pSAK105,5.8kb), and their Pstl restnction patterns were very similar. One band had an identical mobility in all the colonies examined (830bp fragment; Fig. 1, lanes 2-6). MIniprep DNA was prepared from representative clones having plasmids in each of these size classes, and then was used to transform DH5a. Designated transformants from each size class were shown to be MMS^, demonstrating that plasmid DNA, not a chromosomal mutation, was responsible for the MMS phenotype of the clones. The 830 bp fragment isolated from plasmid pSAKIOI was hybridized with plasmid DNA from the five MMS" clones digested with EcoRI-H/ndlll restriction enzymes. Analysis of the resulting autoradiograph revealed that each of the EcoRI-H/ndlll fragments had homology with the 830bp fragment of plasmid pSAKI 01 (Fig. 1, lanes 8-12). These data suggest that all of the cloned inserts encompass the same portion of the chromosome that has rec-A-like activity.
Single and double digestions of plasmid pSAKIOI with a number of restriction enzymes were used to construct a restriction map of piasmid DNA from the RecA clone (Fig. 3). Studies were done to localize the B. pertussis recA gene sequence within the 3.5kb EcoRI-H/ndlll fragment by isolating a number of deletion derivatives of this insert. Different mutants containing deleted plasmids pSAK110, pSAK114, and pSAK115 all became RecA", while a mutant containing plasmid pSAK120 with a single kilobase deletion from a H/ndlll-Sful fragment retained the RecA^ phenotype. The results (Fig. 3) clearly demonstrate that the B. pertussis recA gene is localized within the 2.5kb Srul-EcoRI fragment.
Resistance of E. coli clones containing pSAK10i tc DNA-damaging agents Qualitative experiments had shown that all of the E. coli clones carrying the B. pertussis recA gene were MMS"^. To quantify these P3sults, £ coli strain HB101 (pSAKIOI), together with control strains RR1(pUC18) (RecA+) and HB101(pUC18) (RecA") were plated onto agar plates supplemented with ampicillin and MMS (0-8mM), The results (Fig. 4A) show that strain RR1(pUC18) grew on plates supplemented with 8mM MMS, and HB101 (pUC18) did not grow on plates containing more than 3mM MMS. HBIOI(pSAKIOI) grew on plates containing MMS concentrations in the same range as RR1(pUC18),
Cloning of the recA gene from Bordetella pertussis
1 2 3 4 5 6 7 8 Kb -8.4
-5.9
Fig. 2. Aijtoradiograph resulting from Southern hybridization ot the 3.5kb EcoRI-Hindlll pSAKIOI insert with genomic DNA from Bordetella species, Genomic DNA trom B, pertussis (lanes 1-3), S, bronchiseptica (lanes 4-5), 8. parapertussis (lane 6), and B, avium (lanes 7-8) was digested with Cla\ restriction endonuclease and subjected to agarose gel electrophoresis on an 0,8% agarose gel. The DNA fragments were transferred to Gene Screen Plus and hybridized to pP)-hexanucleotidelabeded 3.5kb ecoRI-Hindlll pSAKIOI insert DNA. Lanes; 1, BP338; 2, BP504; 3, BP165, 4. 64C; 5, B205; 6. 230; 7, 197; 8, 838,
but at approximately 66% of the level attained by RR1 (pUC18). These results demonstrate that HB101 (pSAKIOI) has an intermediate level of resistance to MMS, relative to the RecA"^ and RecA" control strains. Experiments were also performed to determine the resistance of the previously described E. coli strains to various fluenoes of u.v. radiation (0.8J M"^. The results (Fig. 4B) show that strain RRI (pUC18) grew on plates after exposure to u.v. light over a wide range of exposure times (up to 120s), and HB101(pUC18) did not survive a 40s exposure period. HB101(pSAK101) grew on plates after exposure to u.v. light over the same range as RR1 (pUCI 8), but at approximately 66% of the level attained by RRI (pUC18). These results demonstrate that HB101 (pSAK101) is resistant, to an intermediate degree, to u.v. exposure as compared with RR1(pUC18) and HB101 (pUC18). The two survival curves (Fig. 4) show that the B. pertussis recA gene is able to restore resistance to the effects of DNA-damaging agents to E. coli RecA" strains.
Detection of B. pertussis recA promoter activity To deteot the possible existence of a recA promoter sequence in plasmid pSAKIOI, the EcoRI-H/ndlll insert was oloned into plasmid pUC19, giving the cloned gene a
1167
reverse orientation (pSAKIII) relative to that found in pSAKIOI. Representative colonies were then picked, purified, and streaked onto agar plates supplemented with MMS. All 10 colonies tested grew well on this media. To quantify these results, dilutions of cultures of DH5a (pSAKIOI), DH5a(pSAKi11), DH5a(pUC18), and DH5a(pUC'(9) were plated onto LB agar plates with and without MMS. After 24 h incubation, the colonies were enumerated, and the percent survival determined. The results (Table 1) indicate that pSAKIOI and pSAK111 promote approximately the same level of protection against DNA damage from MMS (85.9% and 83.3% survival, respectively), suggesting that the B. pertussis recA gene can be expressed in both orientations. These data strongly suggest that the B. pertussis recA promoter is present in the cloned insert of pSAKI 01.
Characterization of the cloned B. pertussis reoA gene product by T7 polymerase/promoter system After the pSAK101 insert was cloned into pT7-5 and pT7-6 generating pSAK116 and pSAK117, respectively, the approximate molecular weight of the S. pertussis recA protein was determined using the procedure of Tabor and Richardson (1985). The results (Fig. 5, lane 5) demonstrate
HSpPS
pSAKIOI
S
PSI
I
II
PSX
SpS
Y '
RecA E
pSAKHO PSAK114
PSAK115 PSAK120
kb
3 J
3.5 I
Fig. 3. Restriction map of pSAKIOI and its derivatives. The approximate limits of the S, pertussis chromosomal DNA fragment from plasmid pSAKIOI, which expresses RecA* activity, and inserts from subclones that are deficient in this activity are shown. Deleted derivatives of pSAKIOI were produced by treatment with restriction endonucleases and subsequent religation of the deleted plasmid. The ability of the resulting clones to complement E. coli RecA" activity was determined, and their RecA phenotype is shown in the right-hand column. The arrow indicates the direction of transcription determined using the T7 polymerase/promoter system of Tabor and Richardson (1985), Symbols: E, EcoRI; H, H/ndlll; P. Psfl; S, Sa/I; Sp, Sp/il; St, Sful; X, Xhol
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S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L Friedman
0
1
2
3
4
5
6
MMS (mM)
7
8
9
20
40
60
80
100
Fig. 4. Survival of RecA * and RecA E, coti strains after treatment with (A) MMS and (B), u,v, light, as described in the Expenmenta/procedures. The results represent the averages of data obtained trom two and four Independent experiments, respectively. Symbols: D, RR1(pUC18); A , HB101(pUC18);andO, HB101(pSAK101),
120
UV (sec)
that after treatment with rifampicin, which inhibits transcription from non-T7 promoters, a single T7 poiymerase/ promoter-specific band was detected by autoradiography. Compared with moiecuiar weight standards, the molecular weight of the expressed protein was estimated to be 40000. Although a faint band was detected from expression of the cloned gene in the opposite orientation (Fig. 5, lane 8), the band was aiso present in the uninduced control strain lacking the recombinant plasmid {Fig. 5, iane 1), indicating that this protein was not produced from the B. pertussis recA gene.
Discussion Previous studies (Walker, 1984; Kokjohn and Milier, 1985; Smith, 1988; Setlow et ai, 1988) have indicated that a number of cloned recA genes from different organisms are responsible for resistance to DNA damage resuiting from chemical agents and u.v. light. The results of this investigation show that the cloned 6. pertussis recA gene, isolated by complementing the MMS^ phenotype of an E. ooli RecA" mutant, codes for resistance to a chemical agent (MMS) and u.v. light. Preliminary results from Hfr mating experiments have shown that the B. pertussis recA gene also complements defective general recombination exhibited by £ coli RecA" mutants (S. A. Kuhi et at., manuscript in preparation). Quantitative experiments have revealed that the levels of complementation (resistance to DNA damage) of chemical and u.v. damage in E. coli RecA" strains are approximateiy the same. These data suggest that the functional activities responsible for repair of DNA damage caused by both types of agents are also simiiar. These data are also consistent with a report from Kenyon and Waiker (1980) which demonstrated that, at the doses used, the magnitude and kinetics of u.v. and mitomycin C induction of DNA repair genes fused to laoZ were very similar. Also consistent with these data is the
fact that no mutants have been isoiated that are deficient only in repair of chemically damaged DNA or u.v. lightdamaged DNA, but not both types cf repair. Recent reports (Ennis er at., 1989; Dutreix et ai, 1989; Tessman and Peterson, 1985) have only demonstrated that RecA functions affecting LexA cleavage induced by both chemical and u.v. DNA-damaging agents are separabie genetically from functions that affect phage repressor cleavage and recombination. This report also shows that use of a cloned B. pertussis reoA gene as a probe for detection of homologous genes in Bordetella species revealed that an 8.4 kb homologous Clal fragment is associated with ail Bordeteila species tested, except B. avium, which possesses a 5.9kb homologous fragment. These data mdicaXe that the recA gene \s conserved within Bordetella species. This was anticipated, since previous reports (Kloos et ai, 1981; Musser et ai, 1986) have shown that S. pertussis, B. bronchiseptica, and B. parapertussis are very closely related. The suggestion has even been made that these organisms cannot be separated into distinct species (Musser ef ai, 1986). Our finding that B. avium has a different size of Cla I fragment that is homologous to the S. pertussis recA fragment is consistent with previous reports (Jackwood et al., 1986; Kersters et ai, 1984;
Table 1. Survival after MMS treatment of E. cof/strains containing the S. pertussis recA gene cloned in both orientations. Strain DH5«(pUC18) DH5n(pUC19) DH5Q(PSAK101) DH5Q(PSAK1 11)
Survival" 15.7 17.7 85.9 83.3
a. Survival was computed by comparing the number of colonies resulting after 24 h growth on LB agar plates with and without 0.928 mM MMS, The results are the averages of two independent experiments.
Cloning of the recA gene from Bordetella pertussis Kd
1
2
3
4
5
6
7
8
206100-
68 -
42 -
25-
Fig. S. Detection of the 8. pertussis RecA protem. £. ooii K38(pGP1-2) alone (lanes 1-2), containing pSAKI 16 (lanes 3-5), or containing pSAKn7 (6-8) was grown in M9 medium supplemented with thiamine (20 Jig ml ')and 18 amino actds (0,1%, minus cysteine and methionine), and the RecA protein was visualized as described in the Experimental prooedures. Lanes: 1, 3, and 6, uninduced; 2, 4, and 7, induced; 5 and 8, induced plus rifampicin.
Musser et al., 1986), which have shown divergence of S. avium from the other Bordetelia species. Using deletion-mapping experiments, the S. pertussis recA gene was localized to a 2.5kb Stu\-EcoV{\ fragment. When the Tabor and Richardson (1985) system was utilized, the B. pertussis recA gene synthesized a protein calculated to be 40 kD. This molecular size is consistent with the range of sizes (about 35-40 kD) of RecA proteins produced from other organisms' cloned recA genes (Smith, 1988). The transcriptional direction of the B. pertussis recA gene was also determined to be from the EcoRI to the H/ndlll site, because only the EcoRI-H/ndlll orientation of the insert (i.e. with the EcoRI site immediately 3' to the T7 promoter) produced the 40kD protein. The wrgene produces a positive regulatory factor that is frans-aoting and necessary for expression of a number of virulence determinants of B. pertussis (Weiss and Falkow, 1984). It is also necessary for expression of these determinants under certain conditions in £ coli. Our data suggest that the B. pertussis recA gene is being expressed in E. coli in the absence of the B. pertussis wrgene product. This contrasts with a study of B. pertussis virulence determinant FHA (Stibitz ef a/., 1988), whioh has shown that this determinant is not expressed without the presenoe of wrin E. coiL The fact that the B. pertussis recA gene cloned in both orientations into pUC vectors complements the MMS^ of E. coli ReoA" strains strongly suggests that the RecA promoter is present in the cloned sequence. Some studies
1169
(Black and Falkow, 1987; Brownlie er ai., 1988) have shown that virulence genes, in the absence of vir, cloned proximal to a strong promoter, oan only be expressed in one orientation in E. coli. In this study, however, the S. pertussis recA gene was expressed in both orientations in E. coli, suggesting that expression of the gene from a strong E coil promoter does not explain the expression of the S. pertussis recA gene in E. ccli. The cloning and initial characterization of the B. pertussis recA gene, reported in this paper, will allow studies to be done concerning the expression of a housekeeping gene of B. pertussis. Data from this study have demonstrated that this gene does not require the presenoe of the v/rgene for expression in E coii. This finding is consistent with the report of Melton and Weiss (1989), who demonstrated that the recA gene is not wr-regulated in S. pertussis. The only other study of a housekeeping gene in B. pertussis was performed with the aroA gene (Maskell et ai., 1988). It was cloned, DNA-sequenced, and expressed in E. coii without the presence of vir. Experiments are in progress to construct and characterize a B. pertussis ReoA" mutant that will facilitate the performance of further studies dealing with the expression of the recA gene. These and other inquiries will permit the mechanism(s) of ReoA function in S. pertussis to be elucidated, and may also clarify the mechanism of pathogenesis of the organism.
Experimental procedures Bacterial strains, plasmids, and media The bacterial strains and plasmids used in this investigation are described In Table 2. All strains of E. coti were grown in LB-broth and manipulated using standard procedures (Maniatis ef a/., 1982). Stock cultures of E. co//were stored in LB-broth containing the appropriate antibiotic and 15% glycerol at -SCC. The media were supplemented with antibiotics at the following concentrations: ampicillin (sodium salt). 100M,g ml ^ and kanamycin sulphate, 40M,g ml '. For DNA isolation, S. pertussis, B. parapertussis, and S. bronchiseptica strains were cultured on Bordet-Gengou agar (Bordet and Gengou, 1906) plates incubated at 37°C. S. avium strains were grown on SSM-S media (Gentry-Weeks ef a/., 1988) at 37°C. All Bordeteffa stock cultures were stored at -8Q°C in liquid Stainer-Scholte medium (Stainer and Schotte, 1970) containing 25% glycerol.
Chemical reagents and enzymes Methyl methanesulphonate (MMS), hexadecyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulphate {SDS), formamide. dextran sulphate, herring-sperm DNA, general grade agarose, and all antibiotics used were purchased from Sigma Chemical Company. Restriction endonucleases, molecular biology grade agarose (GTG), DH5a competent cells, and T4 DNA ligase were purchased from Bethesda Research Laboratories.
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S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L. Friedman
Table 2. Bacterial strains and plasmids.
Strain/Plasmid
Genotype/Phenotype
Source/Reference
Prototrophic Prototrophic Prototrophic
C. Mandark^ C. Manciark C. Manciark
Prototrophic Prototrophic
A. Bertschinger" A. Bertschinger
Prototrophic
Per Askeloff"
Prototrophic Prototrophic
Gentry-Weeks etai. (1988) Gentry-Weeks etai. (1988)
RRl DH5u
hsdS2Q hsdRU, endA^.
Maniatis efa/, (1982) BRL
HB101 K38
hsdS20. recA^3 HfrC (lambda)
Maniatis efa/, (1982) Russel and Model (1984)
Ap". map • Ap" Ap" Ap". recA' Ap", recA" Ap". recA* Ap". recA' Ap". recA' Ap". recA Ap", recA * Ap", recA Ap". reoA Ap", recA' Ap", recA' Ap", reoA
Tabor and Richardson (1985) Tabor and Richardson (1985) Tabor and Richardson (1985) This study This study This study This study This study From pSAKIOI This study From pSAKIOI From pSAKIOI This Study This study From pSAKIOI
S, pertussis BP338 BP504 BP165 S, bronchiseptica 64C B205 B. parapertussis 230 B. avium 197 838 E. coii
Plasmids pGP-1 pT7-5 pT7-6 pSAKlOO pSAKIOI PSAK102 PSAK104 PSAK105 pSAKIlO pSAK111 PSAK114 pSAK115 PSAK116 pSAKn7 pSAK120
a. United States Food and Drug Administration, Bethesda, MD, USA, b. Veterinary Institute, University of Zurich, Zurich, Switzerland. c. National Bacteriology Laboratory, Sweden,
DNA polymerase I (Klenov/fragment) and calf-intestinal phosphatase were bought from Promega. A hexanucleotide-labelling kit was purchased from Boehringer Mannheim. n-p^P]-dCTP v^'as purchased from Dupont NEN Research Products, and X-AR5 autoradiographic film was purchased from Eastman Kodak. Gene Clean kits were bought from Bio 101.
Recombinant DNA techniques Most molecular and recombinant techniques were performed using the procedures of Maniatis ef a/. (1982). Digestion with restriction enzymes was done using the manufacturer's instructions. Routine subcloning of DNA was completed according to directions supplied v*(ith the Gene Clean kit (Bio 101), and larger concentrations of DNA fragments utilized for cloning were purified from agarose gels by using the Elutrap apparatus of
Schleicher and Schuell according to the manufacturer's instructions. Preparation of competent cells and transformation of E. coti strains were performed according to a modified Hanahan protocol (Kushner, 1978). Radiolabelling of DNA probes employed u-p^Pl-dCTP (3000Ci mmol"') and used Ihe randomhexamer-labelling protocol supplied in a kit from Boehringer Mannheim, Southern transfer and hybridization were done using Gene Screen Plus according to the directions supplied by Dupont NEN Research Products, Southern transfer was accomplished using a Vacublot vacuum blotting apparatus (American Bionetics), and hybridization and washing were accomplished by employing an Omniblot hybridization apparatus (American Bionetics) according to the instructions supplied by the respective manufacturers. Pre-hybridizations and hybridizations were performed at 42''C in a mixture containing 50% tormamide, 1 % SDS, 10% dextran sulphate. 6 x SSC (1 x SSC = 0.15M sodium chloride (NaCI) + 0.015M sodium citrate), and lOOjig m l '
Cioning of the reoA gene from Bordetella pertussis denatured herring-sperm ONA, Post-hybridization final washes were done using a solution containing 0.1 x SSC and 0.1 % SDS at 65°C.
Preparation of genomic DNA frcm Bordetella strains, and construction of a B. pertussis iibrary Chromosomal DNA was prepared from all Bordeteiia strains by a CTAB/NaCI extraction procedure as detailed by Ausubel et aL (1988). The genomic library of B. pertussis BP504 constructed in plasmid vector pUC18 (Yanisch-Perron et ai. 1985) was kindly provided by Dr Kenneth Ryan. Briefly, the genomic DNA was partially digested with Sau3A, and the resulting chromosomal fragments were size-fractionated by density gradient centrifugation in a 10-40% sucrose gradient employing an SW27 ultracentrifuge rotor (Beckman Instruments) according to Maniatis el ai (1982), Chromosomal fragments between 3 and 10kb in length were recovered, dialysed, and ligated into the SamHI site of pUC18 using standard methods (Maniatis ef al., 1982), The ligation mixture was then used to transform DH5a, selecting for Ap" colonies (Kushner, 1978).
MMS and u.v. resistance determinations Quantitative MMS resistance was determined by spread-plating dilutions of bacterial cultures (1-2 x 10° cells ml ') on LB agar media containing various concentrations of MMS, The plates were incubated for 24h at 37°C. and the colonies enumerated. Individual clones were tested for MMS"^ by streaking a portion of an overnight culture for isolation on LB agar plates that contained MMS at a final concentration ot 0.928mM, Resistance to u.v. light was determined by employing cells grown in Hershey broth (Steinberg and Edgar, 1962) to a concentration of 1-2 x 10^ cells ml '. A 5 ml aliquot ot cells was pelleted (4000 x g for 5 min at 4°C), resuspended in an equal volume of M9 broth (Maniatis ef al., 1982). and kept on ice until irradiation. Irradiation was performed with a 1.8ml aliquot of the celts in a lOx 35mm Petridisti. The dish containing the cells was gently swirled during irradiation from a GE Germicidal lamp (General Electric) with an aperture size allowing delivery of 0.8J M ^ s 'for various time intervals. Determination of cell survival under conditions that prevent photoreactivation was done by plating dilutions of the irradiated cells on Hershey agar plates, using the procedure of Adams (1959). Colonies were enumerated after incubation tor 18h at 37°C.
Detection cf RecA gene product Detection of the S, pertussis RecA protein was performed by using the T7 polymerase/promoter system of Tabor and Richardson (1985), This procedure is based on preferential transcription/ translation of genes cloned 3' to a T7 promoter. The translation products resulting from this reaction were resolved on a polyacrylamide gel. and the T7 polymerase/promoter product(s) were detected by autoradiography.
Acknowledgements The authors wish to thank Harris Bemstein and Michael Moran for critically reviewing the manuscript, Diana Humphreys for secreta-
1171
rial assistance, and Michelle Harris for technical assistance. This work was supported by Public Health Service Grant R01A122822 to R.L.F, trom the Nationa) Institute of Allergy and Infectious Diseases, the National Institute of Health, and a Young Investigators Grant from the National Foundation for Infectious Diseases and Biomedicai Research Support Grant 2S07 RR05675-21 to S.A.K.
References Adams, M.H. (1959) Bacteriophages. New York; Interscience Publishers, Inc. Ausubel, F.M., Brent, R,, Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J,A., and Struhl, K. (1988) Current Protocois in Moiecuiar Bioiogy. New York: John Wiley and Sons. Inc. Black, W,J., and Falkow, S. (1987) Construction and characterization of Bordetella pertussis toxin mutants. Infect Immun 55: 2465-2470, Bordet, J., and Gengou, O. (1906) Le microbe de la coqueluche. Ann inst Pasteur 20: 73-\-741. Brownlie, R.M., Coote, J.G., Parton, R,, Schultz, J.E., Rogel, A., and Hanski, E. (1988) Cloning of tne adenylate cyclase genetic determinant of Bordeteiia pertussis and its expression in Escherichia co//and 6. pertussis. hAicrobial Pathoi^: 335-344. Dutreix, M., Moreau, PL., Bailone, A,, Galibert, F., Battista, J.R,, Walker, G.C, and Devoret, R, (1989) New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence tor an additional role tor RecA protein in UV mutagenesis, J Sacfer/o/171: 2415-2423. Ennis, D,G., Ossanna, N., and Mount, D.W. (1989) Genetic separation of Escherichia coii recA functions for SOS mutagenesis and repressor cleavage. J Bacteriol 171: 25332541, Gentry-Weeks, C.R., Cookson, B.T., Goldman, W.E., Rimler. RB., Porter, S.B., and Curtiss III, R. (1988) Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordeteiia avium. Infect immun 56: 1698-1707. Horii, T., Ogawa, T., and Ogawa, H. (1980) Organization of the recA gene of Escherichia coli. Proc NatI Acad Sci USA 77: 313-317. Jackwood, M,W., Sasser, M., and Saif, Y.M. (1986) Contribution to the taxonomy of the turkey coryza agent: cellular fatty acid analysis of the bacterium. Avian Dis 30: 172-178, Kenyon, C.J,. and Walker, G.C. (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc NatiAcad Sci USA 77: 2819-2823. Kersters, K.. Hinz, K,H,, Hertle, A., Segers. P., Lievens, A., Seigmann, O., and DeLey, J. (1984) Bordeteiia avium sp. nov. isolated from the respiratory tracts of turkeys and other birds. Int J Syst Bacterioi 34: 56-70. Kloos, W.E., Mohapatra, N., Dobrogosz, W.J., Ezzell, J.W., and Manciark, C,R. (1981) Deoxyribonucleotide sequence relationships among Bordetella species. Int J Syst Bacteriol 3 1 : 173-176. Kokjohn, T,A., and Miller, R.V. (1985) Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO. J Bacteriol 163: 568-572. Kushner, S.R. 0978) An improved method for transformation ot Escherichia coii with ColEI-derived plasmids. In Genetic Engineering. Boyer, H., and Nicosia, S. (eds). Amsterdam; Eisevier Biomedicai Press, pp. 17-23.
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Unnemann, CO., Jr (1978) Host-parasite interactions in pertussis. In internationat Symposium on Pertussis. Manclark, C.R., and Hill, J.C. (eds). Bethesda, Maryland: National Institutes of Health, pp. 3-18. Little, J.W, (1984) Autodigestion of lexA and phage lambda repressors, Proc NatI Acad Sci USA 81:1375-1379. Little. J.W., and Mount, D.W. (1982) The SOS regulatory system of Escherichia coii Cell 29:11-22. Maniatis. T , Fritsch, E.F., and Sambrook, J.F. (1982) Molecutar Cloning. A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, Maskell, D.J., Morrissey, P., and Dougan, G. (1988) Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis. J Bacteriol 170: 2467-2471. McLafferty, M.A., Harcus, D.R.. and Hewlett, E.L, (1988) Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence. J Gen Microbiol 134: 2297-2306. McPheat, W.L,, and McNally, T. (1987) Isolation of a repeated DNA sequence from Bordetella pertussis. J Gen Microbiol 133; 323-330. Melton, A.R,. and Weiss, A.A. (1989) Environmental regulation of expression of virulence determinants in Bordetella pertussis. J Bacteriol 171: 6206-6212. Musser, J.M., Hewlett. E.L.. Peppier, M.S., and Selander, R.K. (1986) Genetic diversity and relationships in populations of Bordetella spp. J Bacteriol 166: 230-237. Park, I., Saurin, W.. and Ullmann. A. (1988) A highly conserved 530 base-pair repeated DNA sequence specific for Bordetella pertussis. FEMS Microbiol Lett 52: 19-24. Peterson, K.R., Ossanna, N., Thiiveris, A.T., Ennis, D.G., and Mount, D.W, (1988) Derepression of specific genes promotes DNA repair and mutagenesis in Escherichia coli. J Bacterioi 170:1-4. Roberts, J.W., and Roberts, C.W. (1975) Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc NatI Acad
SciUSA72: 147-151. Roberts, J.W., Roberts, C,W., and Mount, O.W. (1977) Inactivation and proteolytic cleavage of phage lambda repressor in vitro in an ATP-dependent reaction. Proc NatlAcad Sci USA 74: 2283-2287. Russel, M., and Model, P. (1984) Replacement of the ftp gene of Escherichia coli by an inactive gene cloned on a plasmid, J eacferio/159: 1034-1039. Sancar, A., and Rupp, W.D. (1979) Physical map of the recA gene. Proc NatlAcad Sci USA 76: 3144-3148. Sanoar, A.. Stacheiek, C , Konigsberg, W., and Rupp, W.D. (1980) Sequences of the recA gene and protein. Proc NatI Acad Sci 77: 2611-2615.
Setlow, J,K,, Spikes, D., and Griffin, K. (1988) Characterization of the rec-^ gene of Haemophilus intluenzae and behavior of the gene in Escherichia coti. J Bacteriot MO: 3876-3881. Slilaty, S.N,. and Little, J.W. (1987) Lysine-156andserine-119are required for LexA repressor cleavage: a possible mechanism. Proc NatlAcad Sci USA 84: 3987-3991. Smith, G.R. (1988) Homologous recombination in procaryotes. Microbiol Rev 52: 1-28. Stainer, D.W,. and Scholte. M.J. (1970) A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol 63: 211 -220, Steinberg, CM., and Edgar, R.S. (1962) A critical test of a current theory of genetic recombination in bacteriophage. Genetics47'. 187-208. Stibitz, S., Weiss, A,A., and Falkow, S. (1988) Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir. J Bacteriot MO: 2904-2913. Tessman, E.S., and Peterson, P. (1985) Isolation of protease-proficient, recombination-deficient recA mutants of Escherichia coli K-12. J Bacteriol 163; 688-695. Tabor, S., and Richardson, C C . (1985) A bacteriophage T7 RNA potymerase/promoter system for controlled exclusive expression of specific genes. Proc NatI Acad Sci USA 82:1074-1078. Walker. G.C, (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli Microbiol Rev 48: 60-93. Walker, G.C (1985) Inducible DNA repair systems. Annu Rev Biochem 54; 425-457. Walker, G.C. (1987) The SOS response of Escherichia coli In Escherichia coli and Salmonella typhimurium: Cellular and Molecutar Biotogy. Neidhardt. F.C, Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M., and Umbarger, H.E. (eds). Washington, D,C.: American Society for Microbiology, pp. 1346-1357. Weiss, A.A., and Falkow, S. (1984) Genetic analysis of phase change in Bordetetta pertussis, infect tmmun 43: 263-269. Weiss, A.A., Hewlett. E.L., Meyers, GA.. and Falkow, S. (1983) Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun 42: 33-41, Witkin. E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coll. Bactertot Rev 40; 869-907. Yanisch-Perron, C , Vieira, J,, and Messing, J. (1985) Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33;
103-119.
Isolation and characterization of the recA gene of Bordetella pertussis S. A. Kuhl,* R. P. McCreary,^ J. D. Bannan and R. L. Friedman Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, Arizona 85724, USA. Summary This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coii RecA" mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA~ strains to tolerate the DNA-damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5kb 5ful-£coRI fragment, and expression of the gene in E. coli resulted in the production of a 40 kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA-like activity has been isolated and cloned.
Introduction The recA gene is one of a number of genes termed 'housekeeping genes' that are concerned with maintenance of cellular functions. The RecA protein has been extensively studied in Escherichia coli, and its dual regulatory roles in the 'SOS-response' Induced by DNA-damaging agents (Witkin, 1976; Little and Mount, 1982; Walker, 1984; 1985) and homologous recombination (Smith, 1988) have been reviewed recently. Researchers have reported (Little, 1984; Slilaty and Little, 1987) that the RecA protein's role in regulating the SOS-response (to DNA damage) is to bind to the LexA repressor protein and stimulate its autodigestion. Cleavage of the LexA repressor derepresses a large number of operons concerned with coping with DNA damage (Little and Mount, 1982; Walker, 1984; 1987; Peterson et al., 1988). The RecA protein also promotes the cleavage of prophage
Received 22 November, 1989. tPresent address: Department of Biology, US Air Force Academy, iJSAFA/DFB, Colorado Springs, Coiorado 80840, USA. 'For correspondence, Tei, (602) 626 6061; Fax (602) 626 2100.
repressors (Roberts and Roberts, 1975; Roberts et al., 1977; Little, 1984), allowing lytic grovrth of bacteriophage to be initiated. Studies performed on a number of different bacteria, including E co//(Walker, 1984; Kokjohn and Miller, 1985; Smith, 1988; Setlow efa/., 1988), have indicated that the recA gene product is required for induced mutagenesis caused by u.v. light and chemical DNA-damaging agents, recombination proficiency, and induction of some prophage. The E. coli recA gene has been cloned, a restriction map constructed (Sanoar and Rupp, 1979), and the DNA sequence determined by Horii etal. (1980) and Sancar et ai. (1980). These authors have found that the gene is 1059 nucleotide residues long and codes for a 38 kD protein. The recA gene has also been cloned from numerous different bacteria including both Gram-negative and Gram-positive organisms (Smith, 1988). One of our research Interests concerns the study of the genetic and molecular aspects of Bordetella pertussis pathogenesis. B. pertussis is a human pathogen that causes whooping cough, an acute and chronic respiratory disease that primarily affects young children (Linnemann, 1978). The virulence-associated and virulence-regulated genes of this organism have been the subject of many studies (Stibitz et al., 1988; Weiss and Falkow, 1984; Weiss et al., 1983), but few investigations have been performed on the regulation and expression of B. pertussis housekeeping genes. This paper describes studies concerned with the B. pertussis recA gene. It details the isolation and initial characterization of the DNA fragment containing this gene from a genomic library. The data presented demonstrate that the recA gene complements the defective RecA phenotype of £ co//recA" mutants by restoring resistance to the effects of DNA-damaging agents, thus confirming its identity.
Results Isolation of the B. pertussis reoA gene The purpose of this study was to isolate and clone the recA gene from 6. pertussis. E. coli strain DH5a(recA1) is sensitive to MMS-induced DNA damage, and can be used to select for clones that complement the RecA" phenotype. A genomic library of S. pertussis DNA in plasmid pUC18 transformed Into DH5a was spread onto agar
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S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L. Friedman
12 3 4 5 6 7
10
11
12
13
-23.1 -9.4 -6.6 -4.4
2.3 2.0
Fig. 1. Autoradiograph resulting from Southern hybridization of the 830bp Pst\ internal fragment of the pSAKIOI insert with plasmid DNA isolated from MMS-resistant clones. Plasmid DNA from the clones was digested with either f ^ f I (lanes 1-7) or EcoRI and H/ndlll (lanes 8-12) and subjected to agarose gel electrophoresis on an 0.8% agarose gel. The DNA fragments were transferred to Gene Screen Plus and hybridized to pP]-h6xanucleotic)e-labelled 830bp Psfl pSAKIOI internal fragment DNA. Lanes: 1 and 7. pUC18; 2 and 8, pSAKlOO: 3 and 9, pSAK101;4and10, pSAK102;5and11,pSAK104;6and 12, pSAK105; 13, H//1 dill-digested lambda D f ^ markers.
Hybridization ot plasmid pSAKIOI with genomic DNA from different Bordetella species With the recA gene from B. pertussis having been isolated, attempts were made to characterize the gene, using the plasmid with the smallest insert (pSAKI 01). To determine whether plasmid pSAKIOI (no Cla\ sites) would hybridize to a single unique band in Bcrdetelia strains, a ^^P-labelled probe of the pSAK101 insert was hybridized with Cla\digested genomic DNA isolated from strains of 6. pertussis, Bcrdetelia bronchiseptica, Bordetelia parapertussis. and Bordetelia avium. The autoradiograph (Fig. 2, lanes 1-6) clearly demonstrates that each of the S. pertussis, B. parapertussis, and B. brcnchiseptica strains examined possessed a single 8.4kb band, while the S. avium strains possessed a 5.9kb band that was homologous to the pSAKIOI piasmid (Fig. 2, lanes 7-8). Sinoe only a single homologous band was detected in B. pertussis genomic DNA, repetitive sequences are not associated with the B. pertussis recA gene, as was previously found in other regions of the 6. pertussis genome (McPheat and McNally, 1987; MoLafferty etaL, 1988; Park efa/., 1988).
Construction of a restriction map of pSAKIOI
plates supplemented with ampicillin and MMS to select for clones resistant to fVlMS (MMS'^. After 24h, MMS" colonies were found at a frequency of 1.69 x 10"^. When DH5a transformed with pUCI 8 without inserts was plated onto media containing MMS, no colonies were visible after 24h. Analysis of 10 representative colonies revealed that five different MMS'^ clones contained plasmids with different size inserts (pSAKlOO, 4.5kb; pSAKIOI, 3.5kb; pSAK102,5.4kb; pSAK104,6.0kb; pSAK105,5.8kb), and their Pstl restnction patterns were very similar. One band had an identical mobility in all the colonies examined (830bp fragment; Fig. 1, lanes 2-6). MIniprep DNA was prepared from representative clones having plasmids in each of these size classes, and then was used to transform DH5a. Designated transformants from each size class were shown to be MMS^, demonstrating that plasmid DNA, not a chromosomal mutation, was responsible for the MMS phenotype of the clones. The 830 bp fragment isolated from plasmid pSAKIOI was hybridized with plasmid DNA from the five MMS" clones digested with EcoRI-H/ndlll restriction enzymes. Analysis of the resulting autoradiograph revealed that each of the EcoRI-H/ndlll fragments had homology with the 830bp fragment of plasmid pSAKI 01 (Fig. 1, lanes 8-12). These data suggest that all of the cloned inserts encompass the same portion of the chromosome that has rec-A-like activity.
Single and double digestions of plasmid pSAKIOI with a number of restriction enzymes were used to construct a restriction map of piasmid DNA from the RecA clone (Fig. 3). Studies were done to localize the B. pertussis recA gene sequence within the 3.5kb EcoRI-H/ndlll fragment by isolating a number of deletion derivatives of this insert. Different mutants containing deleted plasmids pSAK110, pSAK114, and pSAK115 all became RecA", while a mutant containing plasmid pSAK120 with a single kilobase deletion from a H/ndlll-Sful fragment retained the RecA^ phenotype. The results (Fig. 3) clearly demonstrate that the B. pertussis recA gene is localized within the 2.5kb Srul-EcoRI fragment.
Resistance of E. coli clones containing pSAK10i tc DNA-damaging agents Qualitative experiments had shown that all of the E. coli clones carrying the B. pertussis recA gene were MMS"^. To quantify these P3sults, £ coli strain HB101 (pSAKIOI), together with control strains RR1(pUC18) (RecA+) and HB101(pUC18) (RecA") were plated onto agar plates supplemented with ampicillin and MMS (0-8mM), The results (Fig. 4A) show that strain RR1(pUC18) grew on plates supplemented with 8mM MMS, and HB101 (pUC18) did not grow on plates containing more than 3mM MMS. HBIOI(pSAKIOI) grew on plates containing MMS concentrations in the same range as RR1(pUC18),
Cloning of the recA gene from Bordetella pertussis
1 2 3 4 5 6 7 8 Kb -8.4
-5.9
Fig. 2. Aijtoradiograph resulting from Southern hybridization ot the 3.5kb EcoRI-Hindlll pSAKIOI insert with genomic DNA from Bordetella species, Genomic DNA trom B, pertussis (lanes 1-3), S, bronchiseptica (lanes 4-5), 8. parapertussis (lane 6), and B, avium (lanes 7-8) was digested with Cla\ restriction endonuclease and subjected to agarose gel electrophoresis on an 0,8% agarose gel. The DNA fragments were transferred to Gene Screen Plus and hybridized to pP)-hexanucleotidelabeded 3.5kb ecoRI-Hindlll pSAKIOI insert DNA. Lanes; 1, BP338; 2, BP504; 3, BP165, 4. 64C; 5, B205; 6. 230; 7, 197; 8, 838,
but at approximately 66% of the level attained by RR1 (pUC18). These results demonstrate that HB101 (pSAKIOI) has an intermediate level of resistance to MMS, relative to the RecA"^ and RecA" control strains. Experiments were also performed to determine the resistance of the previously described E. coli strains to various fluenoes of u.v. radiation (0.8J M"^. The results (Fig. 4B) show that strain RRI (pUC18) grew on plates after exposure to u.v. light over a wide range of exposure times (up to 120s), and HB101(pUC18) did not survive a 40s exposure period. HB101(pSAK101) grew on plates after exposure to u.v. light over the same range as RR1 (pUCI 8), but at approximately 66% of the level attained by RRI (pUC18). These results demonstrate that HB101 (pSAK101) is resistant, to an intermediate degree, to u.v. exposure as compared with RR1(pUC18) and HB101 (pUC18). The two survival curves (Fig. 4) show that the B. pertussis recA gene is able to restore resistance to the effects of DNA-damaging agents to E. coli RecA" strains.
Detection of B. pertussis recA promoter activity To deteot the possible existence of a recA promoter sequence in plasmid pSAKIOI, the EcoRI-H/ndlll insert was oloned into plasmid pUC19, giving the cloned gene a
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reverse orientation (pSAKIII) relative to that found in pSAKIOI. Representative colonies were then picked, purified, and streaked onto agar plates supplemented with MMS. All 10 colonies tested grew well on this media. To quantify these results, dilutions of cultures of DH5a (pSAKIOI), DH5a(pSAKi11), DH5a(pUC18), and DH5a(pUC'(9) were plated onto LB agar plates with and without MMS. After 24 h incubation, the colonies were enumerated, and the percent survival determined. The results (Table 1) indicate that pSAKIOI and pSAK111 promote approximately the same level of protection against DNA damage from MMS (85.9% and 83.3% survival, respectively), suggesting that the B. pertussis recA gene can be expressed in both orientations. These data strongly suggest that the B. pertussis recA promoter is present in the cloned insert of pSAKI 01.
Characterization of the cloned B. pertussis reoA gene product by T7 polymerase/promoter system After the pSAK101 insert was cloned into pT7-5 and pT7-6 generating pSAK116 and pSAK117, respectively, the approximate molecular weight of the S. pertussis recA protein was determined using the procedure of Tabor and Richardson (1985). The results (Fig. 5, lane 5) demonstrate
HSpPS
pSAKIOI
S
PSI
I
II
PSX
SpS
Y '
RecA E
pSAKHO PSAK114
PSAK115 PSAK120
kb
3 J
3.5 I
Fig. 3. Restriction map of pSAKIOI and its derivatives. The approximate limits of the S, pertussis chromosomal DNA fragment from plasmid pSAKIOI, which expresses RecA* activity, and inserts from subclones that are deficient in this activity are shown. Deleted derivatives of pSAKIOI were produced by treatment with restriction endonucleases and subsequent religation of the deleted plasmid. The ability of the resulting clones to complement E. coli RecA" activity was determined, and their RecA phenotype is shown in the right-hand column. The arrow indicates the direction of transcription determined using the T7 polymerase/promoter system of Tabor and Richardson (1985), Symbols: E, EcoRI; H, H/ndlll; P. Psfl; S, Sa/I; Sp, Sp/il; St, Sful; X, Xhol
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S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L Friedman
0
1
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MMS (mM)
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40
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Fig. 4. Survival of RecA * and RecA E, coti strains after treatment with (A) MMS and (B), u,v, light, as described in the Expenmenta/procedures. The results represent the averages of data obtained trom two and four Independent experiments, respectively. Symbols: D, RR1(pUC18); A , HB101(pUC18);andO, HB101(pSAK101),
120
UV (sec)
that after treatment with rifampicin, which inhibits transcription from non-T7 promoters, a single T7 poiymerase/ promoter-specific band was detected by autoradiography. Compared with moiecuiar weight standards, the molecular weight of the expressed protein was estimated to be 40000. Although a faint band was detected from expression of the cloned gene in the opposite orientation (Fig. 5, lane 8), the band was aiso present in the uninduced control strain lacking the recombinant plasmid {Fig. 5, iane 1), indicating that this protein was not produced from the B. pertussis recA gene.
Discussion Previous studies (Walker, 1984; Kokjohn and Milier, 1985; Smith, 1988; Setlow et ai, 1988) have indicated that a number of cloned recA genes from different organisms are responsible for resistance to DNA damage resuiting from chemical agents and u.v. light. The results of this investigation show that the cloned 6. pertussis recA gene, isolated by complementing the MMS^ phenotype of an E. ooli RecA" mutant, codes for resistance to a chemical agent (MMS) and u.v. light. Preliminary results from Hfr mating experiments have shown that the B. pertussis recA gene also complements defective general recombination exhibited by £ coli RecA" mutants (S. A. Kuhi et at., manuscript in preparation). Quantitative experiments have revealed that the levels of complementation (resistance to DNA damage) of chemical and u.v. damage in E. coli RecA" strains are approximateiy the same. These data suggest that the functional activities responsible for repair of DNA damage caused by both types of agents are also simiiar. These data are also consistent with a report from Kenyon and Waiker (1980) which demonstrated that, at the doses used, the magnitude and kinetics of u.v. and mitomycin C induction of DNA repair genes fused to laoZ were very similar. Also consistent with these data is the
fact that no mutants have been isoiated that are deficient only in repair of chemically damaged DNA or u.v. lightdamaged DNA, but not both types cf repair. Recent reports (Ennis er at., 1989; Dutreix et ai, 1989; Tessman and Peterson, 1985) have only demonstrated that RecA functions affecting LexA cleavage induced by both chemical and u.v. DNA-damaging agents are separabie genetically from functions that affect phage repressor cleavage and recombination. This report also shows that use of a cloned B. pertussis reoA gene as a probe for detection of homologous genes in Bordetella species revealed that an 8.4 kb homologous Clal fragment is associated with ail Bordeteila species tested, except B. avium, which possesses a 5.9kb homologous fragment. These data mdicaXe that the recA gene \s conserved within Bordetella species. This was anticipated, since previous reports (Kloos et ai, 1981; Musser et ai, 1986) have shown that S. pertussis, B. bronchiseptica, and B. parapertussis are very closely related. The suggestion has even been made that these organisms cannot be separated into distinct species (Musser ef ai, 1986). Our finding that B. avium has a different size of Cla I fragment that is homologous to the S. pertussis recA fragment is consistent with previous reports (Jackwood et al., 1986; Kersters et ai, 1984;
Table 1. Survival after MMS treatment of E. cof/strains containing the S. pertussis recA gene cloned in both orientations. Strain DH5«(pUC18) DH5n(pUC19) DH5Q(PSAK101) DH5Q(PSAK1 11)
Survival" 15.7 17.7 85.9 83.3
a. Survival was computed by comparing the number of colonies resulting after 24 h growth on LB agar plates with and without 0.928 mM MMS, The results are the averages of two independent experiments.
Cloning of the recA gene from Bordetella pertussis Kd
1
2
3
4
5
6
7
8
206100-
68 -
42 -
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Fig. S. Detection of the 8. pertussis RecA protem. £. ooii K38(pGP1-2) alone (lanes 1-2), containing pSAKI 16 (lanes 3-5), or containing pSAKn7 (6-8) was grown in M9 medium supplemented with thiamine (20 Jig ml ')and 18 amino actds (0,1%, minus cysteine and methionine), and the RecA protein was visualized as described in the Experimental prooedures. Lanes: 1, 3, and 6, uninduced; 2, 4, and 7, induced; 5 and 8, induced plus rifampicin.
Musser et al., 1986), which have shown divergence of S. avium from the other Bordetelia species. Using deletion-mapping experiments, the S. pertussis recA gene was localized to a 2.5kb Stu\-EcoV{\ fragment. When the Tabor and Richardson (1985) system was utilized, the B. pertussis recA gene synthesized a protein calculated to be 40 kD. This molecular size is consistent with the range of sizes (about 35-40 kD) of RecA proteins produced from other organisms' cloned recA genes (Smith, 1988). The transcriptional direction of the B. pertussis recA gene was also determined to be from the EcoRI to the H/ndlll site, because only the EcoRI-H/ndlll orientation of the insert (i.e. with the EcoRI site immediately 3' to the T7 promoter) produced the 40kD protein. The wrgene produces a positive regulatory factor that is frans-aoting and necessary for expression of a number of virulence determinants of B. pertussis (Weiss and Falkow, 1984). It is also necessary for expression of these determinants under certain conditions in £ coli. Our data suggest that the B. pertussis recA gene is being expressed in E. coli in the absence of the B. pertussis wrgene product. This contrasts with a study of B. pertussis virulence determinant FHA (Stibitz ef a/., 1988), whioh has shown that this determinant is not expressed without the presenoe of wrin E. coiL The fact that the B. pertussis recA gene cloned in both orientations into pUC vectors complements the MMS^ of E. coli ReoA" strains strongly suggests that the RecA promoter is present in the cloned sequence. Some studies
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(Black and Falkow, 1987; Brownlie er ai., 1988) have shown that virulence genes, in the absence of vir, cloned proximal to a strong promoter, oan only be expressed in one orientation in E. coli. In this study, however, the S. pertussis recA gene was expressed in both orientations in E. coli, suggesting that expression of the gene from a strong E coil promoter does not explain the expression of the S. pertussis recA gene in E. ccli. The cloning and initial characterization of the B. pertussis recA gene, reported in this paper, will allow studies to be done concerning the expression of a housekeeping gene of B. pertussis. Data from this study have demonstrated that this gene does not require the presenoe of the v/rgene for expression in E coii. This finding is consistent with the report of Melton and Weiss (1989), who demonstrated that the recA gene is not wr-regulated in S. pertussis. The only other study of a housekeeping gene in B. pertussis was performed with the aroA gene (Maskell et ai., 1988). It was cloned, DNA-sequenced, and expressed in E. coii without the presence of vir. Experiments are in progress to construct and characterize a B. pertussis ReoA" mutant that will facilitate the performance of further studies dealing with the expression of the recA gene. These and other inquiries will permit the mechanism(s) of ReoA function in S. pertussis to be elucidated, and may also clarify the mechanism of pathogenesis of the organism.
Experimental procedures Bacterial strains, plasmids, and media The bacterial strains and plasmids used in this investigation are described In Table 2. All strains of E. coti were grown in LB-broth and manipulated using standard procedures (Maniatis ef a/., 1982). Stock cultures of E. co//were stored in LB-broth containing the appropriate antibiotic and 15% glycerol at -SCC. The media were supplemented with antibiotics at the following concentrations: ampicillin (sodium salt). 100M,g ml ^ and kanamycin sulphate, 40M,g ml '. For DNA isolation, S. pertussis, B. parapertussis, and S. bronchiseptica strains were cultured on Bordet-Gengou agar (Bordet and Gengou, 1906) plates incubated at 37°C. S. avium strains were grown on SSM-S media (Gentry-Weeks ef a/., 1988) at 37°C. All Bordeteffa stock cultures were stored at -8Q°C in liquid Stainer-Scholte medium (Stainer and Schotte, 1970) containing 25% glycerol.
Chemical reagents and enzymes Methyl methanesulphonate (MMS), hexadecyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulphate {SDS), formamide. dextran sulphate, herring-sperm DNA, general grade agarose, and all antibiotics used were purchased from Sigma Chemical Company. Restriction endonucleases, molecular biology grade agarose (GTG), DH5a competent cells, and T4 DNA ligase were purchased from Bethesda Research Laboratories.
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S. A. Kuhl, R. P. McCreary, J. D. Bannan and R. L. Friedman
Table 2. Bacterial strains and plasmids.
Strain/Plasmid
Genotype/Phenotype
Source/Reference
Prototrophic Prototrophic Prototrophic
C. Mandark^ C. Manciark C. Manciark
Prototrophic Prototrophic
A. Bertschinger" A. Bertschinger
Prototrophic
Per Askeloff"
Prototrophic Prototrophic
Gentry-Weeks etai. (1988) Gentry-Weeks etai. (1988)
RRl DH5u
hsdS2Q hsdRU, endA^.
Maniatis efa/, (1982) BRL
HB101 K38
hsdS20. recA^3 HfrC (lambda)
Maniatis efa/, (1982) Russel and Model (1984)
Ap". map • Ap" Ap" Ap". recA' Ap", recA" Ap". recA* Ap". recA' Ap". recA' Ap". recA Ap", recA * Ap", recA Ap". reoA Ap", recA' Ap", recA' Ap", reoA
Tabor and Richardson (1985) Tabor and Richardson (1985) Tabor and Richardson (1985) This study This study This study This study This study From pSAKIOI This study From pSAKIOI From pSAKIOI This Study This study From pSAKIOI
S, pertussis BP338 BP504 BP165 S, bronchiseptica 64C B205 B. parapertussis 230 B. avium 197 838 E. coii
Plasmids pGP-1 pT7-5 pT7-6 pSAKlOO pSAKIOI PSAK102 PSAK104 PSAK105 pSAKIlO pSAK111 PSAK114 pSAK115 PSAK116 pSAKn7 pSAK120
a. United States Food and Drug Administration, Bethesda, MD, USA, b. Veterinary Institute, University of Zurich, Zurich, Switzerland. c. National Bacteriology Laboratory, Sweden,
DNA polymerase I (Klenov/fragment) and calf-intestinal phosphatase were bought from Promega. A hexanucleotide-labelling kit was purchased from Boehringer Mannheim. n-p^P]-dCTP v^'as purchased from Dupont NEN Research Products, and X-AR5 autoradiographic film was purchased from Eastman Kodak. Gene Clean kits were bought from Bio 101.
Recombinant DNA techniques Most molecular and recombinant techniques were performed using the procedures of Maniatis ef a/. (1982). Digestion with restriction enzymes was done using the manufacturer's instructions. Routine subcloning of DNA was completed according to directions supplied v*(ith the Gene Clean kit (Bio 101), and larger concentrations of DNA fragments utilized for cloning were purified from agarose gels by using the Elutrap apparatus of
Schleicher and Schuell according to the manufacturer's instructions. Preparation of competent cells and transformation of E. coti strains were performed according to a modified Hanahan protocol (Kushner, 1978). Radiolabelling of DNA probes employed u-p^Pl-dCTP (3000Ci mmol"') and used Ihe randomhexamer-labelling protocol supplied in a kit from Boehringer Mannheim, Southern transfer and hybridization were done using Gene Screen Plus according to the directions supplied by Dupont NEN Research Products, Southern transfer was accomplished using a Vacublot vacuum blotting apparatus (American Bionetics), and hybridization and washing were accomplished by employing an Omniblot hybridization apparatus (American Bionetics) according to the instructions supplied by the respective manufacturers. Pre-hybridizations and hybridizations were performed at 42''C in a mixture containing 50% tormamide, 1 % SDS, 10% dextran sulphate. 6 x SSC (1 x SSC = 0.15M sodium chloride (NaCI) + 0.015M sodium citrate), and lOOjig m l '
Cioning of the reoA gene from Bordetella pertussis denatured herring-sperm ONA, Post-hybridization final washes were done using a solution containing 0.1 x SSC and 0.1 % SDS at 65°C.
Preparation of genomic DNA frcm Bordetella strains, and construction of a B. pertussis iibrary Chromosomal DNA was prepared from all Bordeteiia strains by a CTAB/NaCI extraction procedure as detailed by Ausubel et aL (1988). The genomic library of B. pertussis BP504 constructed in plasmid vector pUC18 (Yanisch-Perron et ai. 1985) was kindly provided by Dr Kenneth Ryan. Briefly, the genomic DNA was partially digested with Sau3A, and the resulting chromosomal fragments were size-fractionated by density gradient centrifugation in a 10-40% sucrose gradient employing an SW27 ultracentrifuge rotor (Beckman Instruments) according to Maniatis el ai (1982), Chromosomal fragments between 3 and 10kb in length were recovered, dialysed, and ligated into the SamHI site of pUC18 using standard methods (Maniatis ef al., 1982), The ligation mixture was then used to transform DH5a, selecting for Ap" colonies (Kushner, 1978).
MMS and u.v. resistance determinations Quantitative MMS resistance was determined by spread-plating dilutions of bacterial cultures (1-2 x 10° cells ml ') on LB agar media containing various concentrations of MMS, The plates were incubated for 24h at 37°C. and the colonies enumerated. Individual clones were tested for MMS"^ by streaking a portion of an overnight culture for isolation on LB agar plates that contained MMS at a final concentration ot 0.928mM, Resistance to u.v. light was determined by employing cells grown in Hershey broth (Steinberg and Edgar, 1962) to a concentration of 1-2 x 10^ cells ml '. A 5 ml aliquot ot cells was pelleted (4000 x g for 5 min at 4°C), resuspended in an equal volume of M9 broth (Maniatis ef al., 1982). and kept on ice until irradiation. Irradiation was performed with a 1.8ml aliquot of the celts in a lOx 35mm Petridisti. The dish containing the cells was gently swirled during irradiation from a GE Germicidal lamp (General Electric) with an aperture size allowing delivery of 0.8J M ^ s 'for various time intervals. Determination of cell survival under conditions that prevent photoreactivation was done by plating dilutions of the irradiated cells on Hershey agar plates, using the procedure of Adams (1959). Colonies were enumerated after incubation tor 18h at 37°C.
Detection cf RecA gene product Detection of the S, pertussis RecA protein was performed by using the T7 polymerase/promoter system of Tabor and Richardson (1985), This procedure is based on preferential transcription/ translation of genes cloned 3' to a T7 promoter. The translation products resulting from this reaction were resolved on a polyacrylamide gel. and the T7 polymerase/promoter product(s) were detected by autoradiography.
Acknowledgements The authors wish to thank Harris Bemstein and Michael Moran for critically reviewing the manuscript, Diana Humphreys for secreta-
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rial assistance, and Michelle Harris for technical assistance. This work was supported by Public Health Service Grant R01A122822 to R.L.F, trom the Nationa) Institute of Allergy and Infectious Diseases, the National Institute of Health, and a Young Investigators Grant from the National Foundation for Infectious Diseases and Biomedicai Research Support Grant 2S07 RR05675-21 to S.A.K.
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